Epigenetics and Dermatology

fruition.

Section 1

Biological and Historical Aspects of Epigenetics

Outline

Chapter 1 Introduction to Epigenetics

Chapter 2 Laboratory Methods in Epigenetics: Lessons, Challenges, and Windows of Opportunity

Chapter 3 Keratinocyte Differentiation and Epigenetics

Chapter 4 Epigenetics and Fibrosis: Lessons, Challenges, and Windows of Opportunity

Chapter 5 Epigenetic Modulation of Hair Follicle Stem Cells

Chapter 6 Epigenetics and the Regulation of Inflammation

Chapter 7 Malignant Transformation and Epigenetics

Chapter 8 Epigenetic Mechanisms of Sirtuins in Dermatology

Chapter 9 MicroRNAs in Skin Diseases

Chapter 1

Introduction to Epigenetics

Yu Liu and Qianjin Lu,    Department of Dermatology, Second Xiangya Hospital of Central South University, Hunan Key Laboratory of Medical Epigenetics, Changsha, Hunan, PR China

Epigenetic modifications are defined as stable and heritable alterations in gene expression and cellular function without changes to the original DNA sequence. Since 1942, when C.H. Waddington first used the term epigenetics to depict a phenomenon that genetics could not explain, the knowledge in this field has soared and evolved into a branch of science with well-understood molecular mechanisms. The well-recognized epigenetic mechanisms comprise DNA methylation, histone modification, and noncoding RNAs (ncRNAs). DNA methylation is a genetically programmed modification that involves the addition of a methyl group onto the position 5′ of a pyrimidine ring on cytosines (5mC). It is referred to as the fifth base and mediated by DNA methyltransferases (DNMTs). In addition, posttranslational modifications on the tails of core histones can switch a favorable or unfavorable chromatin status for gene transcription. The roles of ncRNAs in regulation of gene expression are sophisticated. However, it is believed that ncRNAs are important epigenetic regulators in cell differentiation, especially microRNAs (miRNAs) and long noncoding RNAs (lncRNAs). With the advancement of technology, a growing body of evidence indicated the role of epigenetics in development and disease. In this chapter, we make an introduction to the function and mediation of the three main epigenetic mechanisms and their association with skin diseases.

Keywords

Epigenetics; DNA methylation; histone modification; microRNAs and long noncoding RNAs; skin diseases

The human genome project has been one of the most important scientific achievements in modern history. It has ushered in a new era in the field of life science research. However, among the project's many great discoveries, surprising findings such as only particular subsets of genes being able to be expressed at a particular location and time, led to the realization that knowledge of DNA sequences is insufficient to understand phenotypic manifestations. The mechanism by which DNA, or the genetic code, is translated into protein sequences is not merely dependent on the sequence itself but also on a sophisticated regulatory system that interplays between genetic and environmental factors. These mechanisms comprise the science of epigenetics, and the control of genes through various chemical interactions for the basis of at least part of the regulatory system overseeing the expression of the genetic code [1].

Epigenetics is defined as heritable changes in gene expression without changes in the DNA sequence. The prefix epi- is derived from the Greek preposition ἐπ , meaning above, on, or over. The term was first coined in 1942 by C.H. Waddington to denote a phenomenon that conventional genetics could not explain [2]. Since then, epigenetics has evolved into a branch of science that studies biological pathways and systems with well-understood molecular mechanisms. Simplistically, epigenetic mechanisms may involve modifications to DNA and surrounding structures such as DNA methylation, chromatin modification, and noncoding RNA (ncRNA).

DNA methylation is a stable and inheritable epigenetic mark. This genetically programmed modification is almost exclusively found on the 5′ position of the pyrimidine ring of cytosines (5mC) adjacent to a guanine. These sites are referred to as CpG sites, and the modification is mediated by specific enzymes called DNA methyltransferases (DNMTs). Transcription is generally repressed by hypermethylation of active promoters associated with CpG-rich sequences [3]. DNA methylation-based imprinting disorders play an important role in skin diseases such as systemic lupus erythematosus (SLE) [4], psoriasis vulgaris [5], primary Sjögren’s syndrome [6], and other diseases. In addition, aberrations in the function of DNMTs and methyl-CpG-binding proteins (MBDs) can also contribute to skin diseases [7]. Recently, another modified form of cytosine, 5-hydroxymethylcytosine (5hmC), has been identified and is now recognized as the sixth base in the mammalian genome, following 5mC (the fifth base) [8]. 5mC can be converted to 5hmC by the ten–eleven translocation (Tet) family proteins, which can further oxidize 5hmC to 5-formylcytosine (5fC) and 5-carboxycytosine (5caC) to achieve active DNA demethylation [9]. Emerging evidence has indicated that 5hmC-mediated DNA demethylation and Tet family proteins may play essential roles in diverse biological processes including development and diseases, as illustrated by the critical function of 5hmC in the development of melanoma [10].

The other main mechanism in epigenetics involves changes to non-DNA gene components. DNA is tightly compacted by histone proteins. Posttranslational modifications on the tails of core histones, including lysine acetylation, lysine and arginine methylation, serine and threonine phosphorylation, and lysine ubiquitination, and sumoylation are important epigenetic modifications that regulate gene transcription. Abnormalities in these modifications, especially acetylation and deacetylation, can alter the structure of chromatin and perturb gene transcription, which can then contribute to disease development and progression. Histone acetylation status is reversibly regulated by two distinct competing families of enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs). Until now, four classes of HDACs have been identified (including Class I, Class II, and Class IV). HDACs are zinc-dependent proteases consisting of HDAC1–11, and Class III, also known as sirtuins (SIRT1–7), which require the cofactor NAD+ for their deacetylase function [11].

Another widely studied histone modification is methylation. Methylation of lysine or arginine in histone proteins alters the compaction or relaxation of chromatin depending on the position of amino acid and the number of methyl groups; for example, histone 3 tri-methylated at lysine 4 promotes gene transcription, while histone 3 tri-methylated at lysine 9 inhibits gene transcription [3]. Increasing evidence indicates the critical role of histone modifications in skin diseases including immune-mediated skin diseases, infectious diseases, and cancer [12–14].

It is debatable whether or not the role of ncRNAs constitutes an epigenetic phenomenon. There are some who will claim that ncRNAs such as microRNAs (miRNAs) are a fundamental part of nature and do not satisfy the definition of epigenetics. However, others feel that since miRNAs do affect regulation of genes, they are a bona fide mechanism of epigenetic change.

The family of ncRNAs is diverse and complex. It can be divided into eight groups: ribosomal RNAs, transfer RNAs, miRNAs, long noncoding RNAs (lncRNAs), small nucleolar RNAs, small interfering RNAs, small nuclear RNAs, and piwi-interacting RNAs. ncRNAs are important epigenetic regulators in development and disease, especially miRNAs and lncRNAs. miRNAs are short ncRNA sequences (19–25 nucleotides) that regulate gene expression by binding to complementary sequences in the 3′ UTR of multiple target mRNAs, leading to translational repression (imperfect sequence match) or mRNA cleavage (perfect match) [15]. Since the first miRNA lin-4 was characterized in 1993, an increasing number of miRNAs have been identified. Altered expression profiles of miRNAs in patients revealed a crucial role of miRNAs in cellular events and the development of diseases [16].

lncRNAs are functional ncRNAs, each exceeding 200 nucleotides in length and lacking functionally open reading frames. lncRNAs regulate gene expression through different molecular mechanisms. They can mediate the activity of proteins involved in chromatin remodeling and histone modification, or act as an RNA decoy or sponge for miRNAs. They can also bind to specific protein partners to modulate the activity of that particular protein [17]. Recent advancements in technology to identify ncRNAs using microarrays provide a great bulk of novel data from genomewide studies, and have revealed potential use of ncRNAs as diagnostic and prognostic biomarkers in various human disorders including skin diseases [18].

The role of genetics in disease is indisputable. But environmental exposures have also been demonstrated to play an essential role in the pathogenesis of skin diseases. Many diseases are now believed to occur as a result of a combination of genetic and environmental factors, but how do these two opposing forces interact? Epigenetic mechanisms may play a role in linking genetic and environmental factors, adding an additional element to the mechanism of disease.

Epigenetic regulation is generally accepted to play a key role in cellular processes. Aberrations of epigenetic modifications contribute to the pathogenesis of human diseases. With a growing knowledge of epigenetic mechanisms, we are confident that epigenetic markers can be applied as sensitive and specific biomarkers in disease diagnosis, evaluation, and prognosis. Moreover, epigenetic interventions may become an important supplement to traditional therapeutic approaches in the near future. The specific role of epigenetics in the pathogenesis, clinical phenotypes, and treatment of skin diseases is rapidly expanding as we continually increase our understanding of the mechanisms of epigenetics.

References

1. Lu Q. The critical importance of epigenetics in autoimmunity. J Autoimmun. 2013;41:1–5.

2. Choudhuri S. From Waddington’s epigenetic landscape to small noncoding RNA: some important milestones in the history of epigenetics research. Toxicol Mech Methods. 2011;21(4):252–274.

3. Liu Y, Li H, Xiao T, Lu Q. Epigenetics in immune-mediated pulmonary diseases. Clin Rev Allergy Immunol. 2013;45(3):314–330.

4. Zhang Y, Zhao M, Sawalha AH, Richardson B, Lu Q. Impaired DNA methylation and its mechanisms in CD4(+) T cells of systemic lupus erythematosus. J Autoimmun. 2013;41:92–99.

5. Zhang P, Zhao M, Liang G, et al. Whole-genome DNA methylation in skin lesions from patients with psoriasis vulgaris. J Autoimmun. 2013;41:17–24.

6. Yu X, Liang G, Yin H, et al. DNA hypermethylation leads to lower FOXP3 expression in CD4+ T cells of patients with primary Sjogren’s syndrome. Clin Immunol. 2013;148(2):254–257.

7. Lei W, Luo Y, Lei W, et al. Abnormal DNA methylation in CD4+ T cells from patients with systemic lupus erythematosus, systemic sclerosis, and dermatomyositis. Scand J Rheumatol. 2009;38(5):369–374.

8. Ye C, Li L. 5-Hydroxymethylcytosine: a new insight into epigenetics in cancer. Cancer Biol Ther. 2014;15(1):10–15.

9. Sun W, Guan M, Li X. 5-Hydroxymethylcytosine-mediated DNA demethylation in stem cells and development. Stem Cells Dev. 2014;23(9):923–930.

10. Lian CG, Xu Y, Ceol C, et al. Loss of 5-hydroxymethylcytosine is an epigenetic hallmark of melanoma. Cell. 2012;150(6):1135–1146.

11. Shi BW, Xu WF. The development and potential clinical utility of biomarkers for HDAC inhibitors. Drug Discov Ther. 2013;7(4):129–136.

12. Trowbridge RM, Pittelkow MR. Epigenetics in the pathogenesis and pathophysiology of psoriasis vulgaris. J Drugs Dermatol. 2014;13(2):111–118.

13. Liang Y, Vogel JL, Arbuckle JH, et al. Targeting the JMJD2 histone demethylases to epigenetically control herpesvirus infection and reactivation from latency. Sci Transl Med. 2013;5(167):167ra5.

14. Rangwala S, Zhang C, Duvic M. HDAC inhibitors for the treatment of cutaneous T-cell lymphomas. Future Med Chem. 2012;4(4):471–486.

15. Hauptman N, Glavac D. MicroRNAs and long non-coding RNAs: prospects in diagnostics and therapy of cancer. Radiol Oncol. 2013;47(4):311–318.

16. Thamilarasan M, Koczan D, Hecker M, Paap B, Zettl UK. MicroRNAs in multiple sclerosis and experimental autoimmune encephalomyelitis. Autoimmun Rev. 2012;11(3):174–179.

17. Katsushima K, Kondo Y. Non-coding RNAs as epigenetic regulator of glioma stem-like cell differentiation. Front Genet. 2014;5:14.

18. Jinnin M. Various applications of microRNAs in skin diseases. J Dermatol Sci. 2014;74(1):3–8.

Chapter 2

Laboratory Methods in Epigenetics

Yu Liu, Jieyue Liao and Qianjin Lu,    Department of Dermatology, Second Xiangya Hospital of Central South University, Hunan Key Laboratory of Medical Epigenetics, Changsha, Hunan, PR China

Epigenetic modifications have been demonstrated to be associated with biological processes through regulation of gene expression. Epigenetic mechanisms mainly consist of DNA methylation, histone modification, and microRNA regulatory activity. DNA methylation involves the addition of a methyl group to the 5′ position of the cytosine pyrimidine ring. DNA hypermethylation generally downregulates gene transcription and the reverse is true for DNA hypomethylation. Histone modification also plays an important role in epigenetic regulation. It is a reversible process, allowing genes to be switched between a favorable or unfavorable chromatin status for transcription. MicroRNAs (miRNAs) are small noncoding RNAs that function as negative gene regulators through binding to complementary sequences in the 3′ UTR of multiple target mRNAs. Here, we describe methods applied in epigenetic studies including technological advances in microarray-based methods and next-generation sequence.

Keywords

Epigenetics; DNA methylation; CpG; 5-hydroxymethylcytosine; histone modification; microRNAs

2.1 Introduction

Epigenetic changes occur during cell differentiation, and serve to activate or suppress genes once the cells have reached terminal differentiation. Thus, epigenetics builds a bridge between genetics and environmental stimuli. Gene expression is up- or downregulated through epigenetic mechanisms in response to environmental changes. Abnormalities of epigenetic marks, such as DNA methylation, histone modifications, and aberrant expression of microRNAs (miRNAs), lead to the development of diseases. Mapping of the human epigenome is one of the most exciting and promising endeavors in terms of increasing our understanding of the etiology of diseases, and of developing new treatment strategies. Recent advances in technology have made it possible to interpret parts of the epigenetic code. In this chapter, we summarize the classical strategies used in epigenetic studies and give a description of technological advancement in detection methodology.

2.2 DNA Methylation Analysis

DNA methylation is an important epigenetic mark and a widely studied epigenetic change. The developments of DNA methylation studies keep pace with the advancements of detection technology. Over the past three decades, a large number of different methods have been applied in DNA methylation analysis. From the initial Southern blot analysis using methylation-sensitive restriction endonucleases to the current availability of microarray-based epigenomics, the technology used for DNA methylation analysis has been revolutionized [1]. Here, we discuss methods to distinguish 5-methylcytosine (5mC) from cytosine as well as methods that can distinguish 5-hydroxymethylcytosine (5hmC) from 5mC. Different methodologies available for analyzing DNA methylation are discussed, with a comparison of their relative strengths and limitations.

2.2.1 Methods to Distinguish 5-Methylcytosine from Cytosine

There are four major methods to distinguish 5-methylcytosine from cytosine. Many additional DNA methylation analysis techniques have been developed based on these primary methods (Figure 2.1).

Figure 2.1 Principles to distinguish 5-methylcytosine from cytosine.

2.2.1.1 Restriction Endonuclease-Based Analysis

2.2.1.1.1 Southern Blot

Southern blot analysis using methylation-sensitive restriction endonucleases is one of the classical and initial methods utilized in the measurement of DNA methylation in particular sequences. The two most commonly used pairs of isoschizomers are HpaII-MspI, which recognize CCGG, and SmaI-XmaI, which recognize CCCGGG. Neither HpaII nor SmaI can digest methylated cytosine [2]. Although this method is relatively inexpensive and the interpretation of results is straightforward, it is limited by the availability of restriction enzyme sites in the target DNA. Other limitations include large amounts of high-quality DNA and problems with incomplete digestions. These disadvantages render this method time-consuming with relatively low resolution. Thus, it is not widely applicable.

2.2.1.1.2 Methylation-Sensitive Amplified Polymorphism

The methylation-sensitive amplified polymorphism (MSAP) method is based on digestion with methylation-sensitive restriction endonucleases followed by amplification of restriction fragments [3]. MSAP is a simple and relatively inexpensive genome-wide method for the identification of putative changes in DNA methylation. Unlike methods based on bisulfite modification or immunoprecipitation, MSAP is independent on the availability of genome sequence information, but the choice of the particular restriction enzymes may lead to ambiguous interpretation of MSAP data [4].

2.2.1.2 Bisulfite Conversion Technique and Derivatives

The bisulfite conversion technique is a revolutionary mark that has accelerated the study of DNA methylation. Treatment of the DNA with sodium bisulfite can convert unmethylated cytosine into uracil, while methylated cytosine remains unchanged. During the following polymerase chain reaction (PCR) process, uracil is then converted to thymidine. This chemical modification in the DNA sequence can be detected by using a variety of methods [5].

2.2.1.2.1 Bisulfite Sequencing PCR

Bisulfite sequencing PCR (BSP), which is regarded as the gold standard of DNA methylation analysis, is an unbiased and sensitive alternative to the use of restriction enzymes. This method combines the bisulfite treatment of genomic DNA with PCR amplification and sequencing analyses [6]. PCR products can be sequenced directly or as single clones. The latter is much more popular as it enables mapping of methylated sites at single-base-pair resolution. To acquire this high-quality data, the bisulfite-treated amplified DNA is usually cloned into bacterial cells with subsequent isolation of plasmids from numerous bacterial clones to be sequenced to determine the extent of methylation within the DNA sequence of interest; this is a process which is quite time-consuming and labor-intensive [7].

2.2.1.2.2 Pyrosequencing

Pyrosequencing is an attractive alternative to the conventional BSP. Pyrosequencing detects luminescence from the release of pyrophosphate on nucleotide incorporation into the complementary strand. Pyrosequencing studies also require the coupling of bisulfite treatment of genomic DNA with PCR amplification of the target sequence, but the advantage of pyrosequencing is that quantitative DNA methylation data can be obtained from direct sequencing of PCR products without requiring cloning into bacterial expression vectors and sequencing a large number of clones [8]. On the other hand, the quality of the data decreases with the distance of the CpG from the 3′ end of the forward primer, thus the number of bases that can be analyzed in a single sequencing reaction is limited [9].

2.2.1.2.3 Combined Bisulfite and Restriction Analysis

Bisulfite treatment of DNA can lead to the creation of new methylation-dependent restriction sites or the maintenance of restriction sites in a methylation-dependent manner. Based on this property, a quantitative method termed combined bisulfite restriction analysis (COBRA) was developed which merged the bisulfite and restriction analysis protocols. The use of COBRA is again limited by the availability of restriction enzyme recognition sites in the target DNA. This method is relatively labor-intensive but is cost-effective [10].

2.2.1.2.4 Methylation-Sensitive Single-Nucleotide Primer Extension and SnuPE Ion Pair Reversed-Phase High Performance Liquid Chromatography

Methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) assay analyzes methylation status at individual CpG sites in a quantitative way and with the capability of multiple analyses. This method couples bisulfite treatment with strand-specific PCR which is performed to generate a DNA template. Subsequently, an internal primer that terminates immediately 5′ of the single nucleotide to be assayed is extended with a DNA polymerase that uses ³²P-labeled dCTP or dTTP [11]. This protocol can be carried out using multiple internal primers in a single primer-extension reaction; thus a relatively high throughput is possible. However, Ms-SNuPE assay is usually labor-intensive and requires radioactive substrates. To overcome this restriction, several variants which omitted radioactive labeling were developed, such as SNaPshot technology from Applied Biosystems (ABI) [12], SNuPE ion pair reversed-phase HPLC (SIRPH) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) [13].

2.2.1.2.5 Methylation-Sensitive Melting Curve Analysis

Based on the principle that the higher GC (base pair of guanine and cytosine) content of DNA sequence makes it more resistant to melting, a new approach to DNA methylation analysis, methylation-sensitive melting curve analysis (MS-MCA), was developed. This method detects sequence difference between methylated and unmethylated DNA obtained after sodium bisulfite treatment by continuous monitoring of the change of fluorescence as a DNA duplex melts while the temperature is increased. If equal proportions of fully methylated and fully unmethylated molecules are amplified, two distinct melting peaks are observed, and interpretation is easy. If the target sequence is heterogeneously methylated, a complex melting will result in a pattern which is difficult to interpret [14].

2.2.1.2.6 Methylation-Sensitive High-Resolution Melting

The principle behind methylation-sensitive high-resolution melting (MS-HRM) is the same as for MS-MCA, but MS-HRM possesses some methodological advantages. First, the HRM approach acquires more data points so that it is more sensitive to detecting subtle differences within the amplicons. Second, the temperature variations produced with HRM instrumentation are generally extremely small. Third, the data obtained in HRM are more stable and reliable because most of the software provided with the instruments allows normalization for end-level fluorescence, temperature shifting, and use of internal oligonucleotide calibrators [14]. This technique requires the use of double-stranded DNA-binding dyes that can be used at saturating concentrations without inhibiting PCR amplification. Both MCA and HRM are semiquantitative measurements that cannot offer detailed information about the methylation of single cytosines within the sequence of interest, but they can distinguish fully and partially methylated samples, which may enable early detection of diseases [15].

2.2.1.2.7 MethyLight

MethyLight technology is a sensitive, sodium-bisulfite-dependent, fluorescence-based real-time PCR technique that quantitatively analyzes DNA methylation. Execution of MethyLight requires the designation of methylation-specific primers and fluorogenic probes [16]. The MethyLight method has major advantages. First, it is a relatively simple assay procedure, without the need to open the PCR tubes after the reaction has ended, thereby reducing the risk of contamination and the handling errors associated with manual manipulation. Second, only small amounts and modest quality of DNA template are required, making the method compatible with plasma samples and small biopsies. Third, it has the potential ability to be used as a rapid screen tool and is uniquely well suited for detection of low-frequency DNA methylation biomarkers as evidence of disease. However, the drawback of MethyLight technology is that it is not designed to offer high-resolution methylation information [17,18].

2.2.1.3 Immunoprecipitation-Based Methods

Immunoprecipitation-based methods utilize methylation-binding proteins such as MeCP2 and MBD2, or 5mC-specific antibodies to enrich the methylated fraction of the genome. Different strategies using this approach have been successfully applied for the analysis of DNA methylation information. The two most commonly used methods are methylated DNA immunoprecipitation (MeDIP) and methyl-CpG immunoprecipitation (MCIp). MeDIP is an adaptation of the chromatin immunoprecipitation (ChIP) technique and uses 5mC-specific antibodies to immunoprecipitate methylated DNA. MCIp uses a recombinant protein that contains the methyl-CpG-binding domain and the Fc fraction of the human IgG1 to directly bind and enrich methylated DNA. These methods are relatively straightforward without either digestion of genomic DNA or bisulfite treatment and the results are relatively easier to analyze and interpret. However, immunoprecipitation-based methods do not provide DNA methylation information at single-nucleotide resolution [19].

2.2.1.3.1 Methylated-CpG Island Recovery Assay

The methyl-CpG island recovery assay (MIRA) is based on the fact that methyl-CpG-binding domain protein-2 (MBD2) has the capacity to bind specifically to methylated DNA sequences and this interaction is enhanced by the methyl-CpG-binding domain protein 3-like-1 (MBD3L1) protein. DNA isolated from cells or tissue is sonicated and incubated with a matrix containing glutathione-S-transferase-MBD2b and MBD3L1. Then, specifically bound DNA is eluted from the matrix and gene-specific PCR reactions are performed to detect CpG island methylation. The MIRA procedure can detect DNA methylation using 1 ng of DNA or 3000 cells. It is quite specific, sensitive, and labor-saving [20].

2.2.1.3.2 Methyl-Binding-PCR

Methyl-binding (MB)-PCR relies on a recombinant, bivalent polypeptide with high affinity for CpG-methylated DNA. This polypeptide is coated onto the walls of a PCR vessel and can selectively capture methylated DNA fragments from a mixture of genomic DNA. Then, the degree of methylation of a specific DNA fragment is detected in the same tube by gene-specific PCR. MB-PCR is particularly useful to screen for methylation levels of candidate genes. Given the enormous amplification capability and specificity of PCR, MB-PCR provides a quick, simple, and extremely sensitive technique that can reliably detect the methylation degree of a specific genomic DNA fragment from <30 cells [21].

2.2.1.4 Mass Spectrometry-Based Methods

Mass spectrometry is recognized as an extremely useful and reliable measurement for acquiring molecular information. The principle of mass spectrometry is that a charged particle passing through a magnetic field is deflected along a circular path on a radius that varies with the mass-to-charge ratio (m/z). One adapted mass spectrometry platform for DNA methylation analysis is MassARRAY EpiTYPER, which uses base-specific enzymatic cleavage coupled to MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight mass spectrometry) mass spectrometry analysis. Although the limited throughput and high cost restrict this approach in becoming a genome-wide technology, it is an excellent tool to analyze DNA methylation for its fast and accurate analysis power and its multichannel analysis capability [22].

2.2.1.4.1 MALDI-TOF Mass Spectrometry with Base-Specific Cleavage

The base-specific cleavage strategy involves amplification of bisulfite-treated DNA followed by in vitro transcription, and subsequent base-specific RNA cleavage by an endoribonuclease to produce different cleavage patterns. Bisulfite treatment of genomic DNA converts unmethylated cytosine into uracil and it appears as a thymidine (T) in the PCR products while the methylated cytosine remains unchanged. These C/T appear as G/A variations in the reverse strand. In the subsequent base-specific RNA cleavage reaction, methylated regions are cleaved at every C to create fragments containing at least one CpG site each. But both methylated and unmethylated regions are cleaved at every T to produce fragments in the T-cleavage reaction. G/A variations in the cleaved products generated from the reverse strand show a mass difference of 16 Da per CpG site. In MALDI-TOF analysis, the relative amount of methylated sequence can be calculated by comparing the signal intensity between the mass signals of methylated and unmethylated templates to generate quantitative results. This approach is recommended for purposes requiring the analysis of larger regions of unknown methylation content [23].

2.2.1.4.2 MALDI-TOF Mass Spectrometry with Primer Extension

The primer-extension strategy requires the designation of a primer that anneals immediately adjacent to the CpG site under investigation in a post-PCR primer-extension reaction. The primer is then extended with a mixture of four different terminators and the extension reaction will terminate on different nucleotides depending on the methylation status of the CpG site. Therefore, distinct signals are generated for MALDI-TOF mass spectrometry analysis. This approach should be used in routine analyses of a relatively small number of well-characterized informative CpG sites [14].

2.2.2 Genome-Scale DNA Methylation Analysis

Given the importance of DNA methylation, it is not surprising that many researchers have taken advantage of array- and sequencing-based technologies that have become available in recent years to perform genome-scale association studies which will provide valuable new information with high throughput and lower cost.

2.2.2.1 Microarray-Based Analysis of DNA Methylation Changes

2.2.2.1.1 Sample Preparation

There are three basic techniques applied to sample preparation in microarray platforms: digesting the DNA with methylation-sensitive or methylation-insensitive restriction endonucleases, sodium bisulfite conversion of unmethylated cytosine into uracil, and affinity purification by applying antibodies binding to methylated cytosines. Coupled with these techniques, a wide range of microarray platforms have evolved to enable genome-scale DNA methylation analysis [22].

2.2.2.1.2 Microarray Used in DNA Methylation Profiling

The initially applied microarray platform was a CpG island microarray used to identify genomic loci that exhibited differential methylation. CGI microarrays used clones from libraries in which CpG-rich fragments had been enriched by MeCP2 columns [24]. However, these arrays have low resolution and limited methylome coverage. Therefore, microarrays made of short oligonucleotides are now commercially available to overcome the drawbacks of CGI microarrays [25]. These oligonucleotide arrays, such as a promoter array, can reach a high resolution, can be easily configured according to the user’s need and often contain a high density of probes spanning each CGI [26]. The first complete high-resolution DNA methylome profile of a living organism (Arabidopsis thaliana) was generated using a tiling array platform [27]. This approach involves up to several million oligonucleotides and has greater methylome coverage than promoter and CGI microarrays. It has allowed researchers to study DNA methylation in noncoding areas in addition to regulatory regions of genes [28,29]. However, to cover the entire human genome, more array slides and a relatively larger amount of genomic DNA are required. The single-nucleotide polymorphism (SNP) arrays combine the use of methylation-specific endonucleases with an SNP-ChIP. This approach can provide an integrated genetic and epigenetic profiling and allows allele-specific methylation analysis at heterozygous loci [30,31]. Besides the methods cited above, microarrays based on methyl-sensitive restriction enzymes, methylation-dependent restriction enzymes, bisulfite conversion, or immunoprecipitation are widely used in epigenomic studies [32]. These microarray-based technologies show differences in terms of resolution, coverage, and sample preparation; therefore, it is necessary to determine the advantages and disadvantages of each specific technique (Table 2.1) [33–41].

Table 2.1

Comparison of Microarray Assays in DNA Methylation Detection

HELP, HpaII tiny fragment enrichment by ligation-mediated PCR; CHARM, comprehensive high-throughput arrays for relative methylation; MeDIP, methylated DNA immunoprecipitation; MIRA, methylated-CpG island recovery assay; CGI shores, stretches of ~2 kb bordering CpG islands.

2.2.2.2 Next-Generation Sequencing Techniques

The evolution of sequencing technologies marked by the first massively parallel DNA sequencing platforms in 2005, has revolutionized research in biological science and ushered in a new era of next-generation sequencing (NGS). There are three major NGS platforms, namely Roche/454, Illumina/Solexa, and Life Technologies/SOLiD, and each of them has different features (Table 2.2) [42–47]. Compared with microarray-based methodologies, NGS offers higher resolution and a larger coverage, and is independent of knowledge of the reference genome or genomic features. Most importantly, NGS methods allow for assessment of DNA methylation in interspersed repetitive genomic regions that are inaccessible using microarrays [22]. However, sequencing-based methods would produce a dramatically large number of bioinformatics data, which leads to extreme difficulties in downstream data management. Thus, the selection of bioinformatics software tools is particularly critical for efficient and appropriate data processing [48]. NGS techniques also include methylation-sensitive restriction enzymes (MRE-seq), affinity-based methods (such as MeDIP-seq, MBD-seq), and bisulfite conversion approaches (e.g., reduced-representation bisulfite sequencing (RRBS)). MRE-seq methods evaluate relative rather than absolute methylation levels through incorporating parallel digestions with three to five restriction endonucleases. With single CpG resolution and the ability to assay a more significant portion of the methylome including most CGIs, MRE-seq becomes a relatively simple and accurate method to analyze DNA methylation [49]. Compared with MRE or bisulfite-based sequencing, MeDIP-seq shows lower resolution, but an important advantage of MeDIP over restriction enzyme methods is that it lacks bias for a specific nucleotide sequence, other than CpGs [49]. RRBS can assess absolute quantification of methylation of more than 1 million CpG sites at single base-pair resolution, which prevails over other sequencing methods [50]. However, all the methods cited above can generate largely comparable methylation calls, but differ in CpG coverage, resolution, quantitative accuracy, efficiency, and cost [51].

Table 2.2

Parameters of NGS Platforms in Profiling DNA Methylome

2.3 Techniques Used for 5hmC Mark Detection

5′-hmC is an oxidative metabolite of 5′mC catalyzed by ten–eleven translocation dioxygenases (TET). It is widely distributed among tissues, especially in embryonic stem cells and Purkinje neurons, but depleted in cancer cell lines, which indicates that 5hmC might serve biologically important roles [52]. Traditional methods for detecting 5mC cannot differentiate 5mC from 5hmC. Methylation-sensitive restriction enzymes are equally blocked by 5mC or 5hmC. Analogously to 5mC, 5hmC remains unconverted after bisulfite treatment [53]. The anti-5mC antibody and the methylated CpG-binding proteins (such as MBD1, MBD2b, MBD3L, MBD4, and the MBD domain of MeCP2) cannot recognize the oxidized base [54]. Therefore, exploration of special methods for 5hmC detection is necessary. To overcome the limitation of traditional bisulfite sequencing, two methods of base-resolution hydroxymethylome mapping were developed: oxidative bisulfite sequencing (oxBS-seq) and Tet-assisted bisulfite sequencing (TAB-seq) (Figure 2.2). The oxidative bisulfite sequencing strategy uses potassium perruthenate (KRuO4) to oxidase 5hmC to 5fC (5-formylcytosine), which could be converted into uracil (U) in the subsequent bisulfite treatment, while 5mC remains as Cs. This allows determination of the amount of 5hmC at a particular nucleotide position by subtraction of this readout from a BS-seq one [55]. The TAB-seq protocol comprises two steps. First, a glucose moiety is introduced onto the hydroxyl group of 5hmC by using β-glucosyltransferase (β-GT) to generate 5ghmC (β-glucosyl-5hmC), in order to prevent further oxidization by Tet1 protein in the next step. After blocking of 5hmC, all 5mC is converted to 5caC (5-carboxylcytosine) by oxidation with excess recombinant Tet1 protein. Then, in the followed bisulfite sequencing, all Cs and 5caCs (from 5mCs) read as Ts, while 5ghmCs (from 5hmCs) are sequenced as Cs. This approach allows for the detection of 5hmC at single-base resolution in both genome-wide and loci-specific studies [56,57]. For genome-wide studies of 5hmC, 5hmC-specific antibodies (hMeDIP) or chemical affinity tags to enrich 5hmC-containing DNA are applied [58–60]. These studies yielded many insightful observations, but were limited by resolution. Recently, Sun et al. reported a genome-wide high-resolution method for hydroxymethylome study which utilizes a 5hmC-dependent restriction endonuclease, AbaSI, that recognizes 5ghmC with high specificity when compared to 5mC and C. This AbaSI-coupled sequencing (Aba-seq) allows researchers to determine the exact 5hmC locations [61].

Figure 2.2 Methods to detect 5-hydroxymethylcytosine.

2.4 Histone Modification Analysis

Modification of histone proteins is an essential component of the regulation of gene activity. Due to the extra complexity and multivariate nature of histone modifications, it has been suggested that the histone modifications may be assimilated in the form of a histone code. Advances have been made in techniques to assess histone modifications on specific residues as well as genome-wide analysis. Of the many assays used to assess the histone modification status, the most fundamental technique is ChIP. The coupling of ChIP with DNA microarray (ChIP-on-chip) and high-throughput sequencing (ChIP-seq) has significantly increased the scope of histone modification analysis.

2.4.1 Chromatin Immunoprecipitation

The ChIP assay is a powerful and versatile technique used for probing interactions between specific proteins or modified forms of proteins and a genomic DNA region. The first use of the ChIP assay was by Gilmour and colleagues in 1984 to monitor the association of RNA polymerase II with transcribed and poised genes in Drosophila cells. Now, this assay is widely used to monitor the presence of particular histone modifications at specific genomic locations. In addition, the ChIP assay can be used to analyze binding of transcription factors, DNA replication factors, and DNA repair proteins [62,63].

When performing the ChIP assay, the initial step is the cross-linking of protein–DNA in live cells with formaldehyde. After cross-linking, cells are lysed and chromatin is harvested and fragmented using either sonication or enzymatic digestion. Proteins together with cross-linked DNA are subsequently subjected to immunoprecipitation using antibodies specific to a particular protein or histone modification. Any DNA fragments that are connected with the protein or histone modification of interest will co-precipitate as part of the cross-linked protein–DNA complexes. After immunoprecipitation, the protein–DNA cross-links are reversed and the DNA is purified. The enrichment of a particular DNA sequence can then be detected by agarose gel electrophoresis or more commonly by quantitative PCR (qPCR). Alternatively, the ChIP assay can be combined with genomic tiling microarray (ChIP-on-chip) techniques, sequencing (ChIP-seq), or cloning strategies, which allow for genome-wide mapping of protein–DNA interactions and histone modifications (Figure 2.3).

Figure 2.3 Overview of chip experiment.

2.4.2 ChIP-on-Chip

ChIP-on-chip is a technique that combines ChIP with microarray technology. In ChIP-on-chip, immunoprecipitated material is labeled with fluorescent dyes and hybridized to DNA microarrays containing several hundred thousand to several million probes [64]. In the first step, ChIP is performed on cross-linked chromatin as described above. After purification and amplification of the DNA, the samples are labeled with a fluorescent tag such as Cy5 or Alexa 647. Labeled IP and input samples are hybridized onto the DNA microarray at 67°C for 24 h. Whenever a labeled fragment finds a complementary fragment on the array, they will hybridize and form a double-stranded DNA fragment. Using the ChIP-on-chip technique, global patterns of different histone modifications can be observed.

The biological significance may be derived from identifying genomic regions where ChIP-on-chip probes show significant enrichment. Often normalized signals and P values are employed for each degree of genomic probe-enrichment. As the microarray has unbiased, high-throughput capabilities, the ChIP-on-chip technique has several significant advantages. First, it allows the identification of histone modification and permits discovery of unanticipated sites of protein-binding DNA on a genome-wide basis instead of a limited number of loci selected by researchers. Second, the ChIP-on-chip technique often has an optimized commercially available platform, and therefore it is time-saving compared with large-scale quantitative PCR assays. Third, the parallel analysis of thousands of genes allows one to parse the data into distinct classes of genes based on different binding distributions or behaviors, and permits statistical comparisons between classes.

2.4.3 ChIP-seq

Owing to the rapid progress in NGS technology, ChIP followed by sequencing (ChIP-seq) can be used to accurately survey interactions between protein, DNA, and RNA.

2.4.3.1 Workflow of ChIP-seq

ChIP-seq typically begins with the process of ChIP and yields several to a few hundred nanograms of DNA as 75- to 300-bp fragments surrounding histone mark locations. High-throughput sequencing is then used to read the ChIP-DNA fragments on a genome-wide scale. The key steps of ChIP-seq are summarized as follows: cross-link protein and shear DNA; add protein-specific antibody; immunoprecipitate and purify complexes; reverse cross-links, purify DNA and prepare for sequencing; sequence DNA fragment and map to